Many techniques are available for the detection of biological materials, for instance by the production of a colour. For example, materials may be added that form hydrogen peroxide or NADH in the presence of the target substance, and these may be caused to produce a coloured compound by further reactions. Alternatively, the target substance may produce colour more directly; for instance, many hydrolytic enzymes can act on artificial substrates to give a coloured product.
A difficulty arises, however, in connection with the detection of very small amounts of the target substance, such that the amount of colour produced may be too small to analyse without complicated apparatus, and certainly not by the naked eye. Some amplification mechanisms have been described, such as cyclic reactions for the measurement of low levels of NAD(H) or NADP(H); see EP-A-0060123. This is an example of a cycle giving linear amplification of the target substances, i.e. their concentrations increase in proportion with reaction time, and therefore a long period is required for sensitive measurements.
GB-A-2055200 discloses the same and other linear amplification cycles. It also discloses a cycle wherein adenylate kinase catalyzes the reaction of AMP and ATP to form ADP which is then re-phosphorylated to form ATP by the action of pyruvate kinase. It is stated that, in each cycle, one extra molecule of ATP is produced. The reaction is used to amplify ATP for the purposes of bioluminescence. The procedure for ATP amplification involves adding the other materials sufficient for the reactions to proceed, stopping the cycling reaction, and measuring accumulated ATP using a bioluminescence assay and a luminometer.
Chittock et al. Biochemical Society Transactions (1991) 19:160S, also disclose that the light intensity of the known bioluminescence reaction may be increased by recycling ATP in a system containing myokinase (adenylate kinase), pyruvate kinase, phosphoenolpyruvate and AMP. The authors consider the fate of two molecules of ATP present initially in the sample. One molecule is consumed by the luciferase reaction and is converted to AMP. It can then undergo a reaction with the other molecule of ATP, in a reaction catalyzed by myokinase (adenylate kinase), to form two molecules of ADP. These are subsequently re-phosphorylated by reaction with phosphoenolpyruvate, catalysed by pyruvate kinase, to regenerate the original two molecules of ATP. The added AMP is described as a "trap" for ATP which helps to get the recycling reaction underway.
It is not clear from the reaction scheme proposed by Chittock et al why the proposed ATP recycling mechanism should cause the observed increase in peak light output of the luciferase reaction, since no net increase in ATP concentration would occur. Nevertheless, the authors describe the phenomenon as amplification of ATP.
WO-A-9525948 discloses a sample-collecting and assay device comprising a tube, a removable top part and a bottom part, wherein an elongate member with a swab at its distal end is mounted on the top part. There may be one or more foil-sealed bottom parts fixed within the tube, and the foils are successively broken by movement of the swab, or a coaxially-extending blade-like member, through the tube.
WO-A-9703209 discloses a similar device, in which reagent is contained in a bottom part with a window, and which has a seal broken by movement of the swab through the tube. Another, similar device is disclosed in WO-A-9723596.